JP6635052B2 - Structured illumination microscope and observation method - Google Patents

Description

  The present invention relates to a structured illumination microscope, an observation method, and a control program.

  2. Description of the Related Art Among microscope devices, there is a super-resolution microscope that enables observation beyond the resolution of an optical system. As one form of this super-resolution microscope, a structured illumination microscope (SIM :) that illuminates a sample with structured illumination to obtain a modulated image, and demodulates the modulated image to generate a super-resolution image of the sample. Structured Illumination Microscopy) is known (for example, see Patent Document 1). In this method, a light beam emitted from a light source is split into a plurality of light beams by a diffraction grating or the like, and the sample is illuminated with interference fringes formed by causing the light beams to interfere with each other in the vicinity of the sample. Are obtained.

US Reissue Patent Invention No. 38307

  Incidentally, the modulated image captured by the structured illumination microscope includes light (signal) from a region within the depth of focus and light from a region outside the depth of focus. Since the contrast of the structured illumination of the image component due to light from an area outside the depth of focus is low, the contrast of the structured illumination in the captured image increases as the thickness of the sample exceeds the depth of focus of the imaging optical system. For example, depending on the device characteristics of the image sensor, it becomes difficult to secure the level of the signal used for demodulation. The present invention has been made in view of the above circumstances, and provides a structured illumination microscope, an observation method, and a control program capable of generating a super-resolution image for a sample having a thickness exceeding the depth of focus of an imaging optical system. The purpose is to do.

According to an aspect of the present invention, there is provided a first illumination optical system for irradiating a sample with activation light for activating a fluorescent substance contained in a sample from a first direction, and an objective lens. A second illumination optical system that irradiates the sample with excitation light for exciting the fluorescent substance from the second direction through the objective lens with interference fringes, a control unit that controls the direction and phase of the interference fringes, An imaging optical system having an objective lens and forming an image of the sample irradiated with the interference fringes; an imaging device for capturing an image formed by the imaging optical system to generate an image; and an imaging device generated by the imaging device. Using a plurality of images, a demodulation unit that generates an image of the sample, comprising: a second illumination optical system, a branching unit that branches the excitation light into a plurality of diffracted lights by diffraction, of the plurality of diffracted lights, A shield capable of switching between a passing state and a blocking state of the + 1st-order or -1st-order diffracted light. Comprising a part, and is structured illumination microscope, wherein provided that.
Further, according to an aspect of the present invention, by irradiating the sample with activation light for activating a fluorescent substance contained in the sample from a first direction, and by using a second illumination optical system having an objective lens, Irradiating the sample with excitation light for exciting the fluorescent substance from the second direction different from the first direction with the interference fringes through the objective lens, controlling the direction and phase of the interference fringes, Forming an image of a sample irradiated with interference fringes by using an imaging optical system having an image, capturing an image to generate an image, and using a plurality of images generated by the imaging to image the sample. And, by the second illumination optical system, the excitation light is split into a plurality of diffracted lights by diffraction, and among the plurality of diffracted lights, the + 1st-order diffracted light or the −1st-order diffracted light is switched between a passing state and a blocking state And including Observation method is provided that.
According to an aspect of the present invention, there is provided a first illumination optical system for irradiating a sample with activation light for activating a fluorescent substance contained in a sample from a first direction, and an objective lens. A second illumination optical system that irradiates the sample with excitation light for exciting the fluorescent substance from the second direction through the objective lens with interference fringes, a control unit that controls the direction and phase of the interference fringes, An imaging optical system having an objective lens and forming an image of the sample irradiated with the interference fringes; an imaging device for capturing an image formed by the imaging optical system to generate an image; and an imaging device generated by the imaging device. There is provided a structured illumination microscope comprising: a demodulation unit that generates an image of a sample using a plurality of images.
According to the aspect of the present invention, the sample is irradiated with activating light for activating the fluorescent substance contained in the sample from the first direction, and the first illumination optical system having the objective lens is used for the first illumination. Irradiating the sample with excitation light for exciting the fluorescent substance from the second direction different from the direction through the objective lens with interference fringes, controlling the direction and phase of the interference fringes, and having the objective lens Forming an image of a sample irradiated with interference fringes by an imaging optical system, capturing an image to generate an image, and generating an image of the sample using a plurality of images generated by the imaging And providing an observation method.
According to an aspect of the present invention, the computer irradiates the sample with the activating light for activating the fluorescent substance contained in the sample from the first direction, and uses the second illumination optical system having the objective lens. Irradiating a sample with excitation light for exciting a fluorescent substance from a second direction different from the first direction through an objective lens with interference fringes, controlling the direction and phase of the interference fringes, An imaging optical system having a lens forms an image of a sample irradiated with interference fringes, captures an image to generate an image, and uses a plurality of images generated by the imaging to form a sample. There is provided a control program for generating an image and causing the program to execute.
According to the first aspect of the present invention, a first illumination optical system that irradiates a sample with activation light for activating a fluorescent substance included in a sample from a first direction, and a first illumination optical system that is different from the first direction. A second illumination optical system that irradiates the sample with excitation light for exciting the fluorescent substance from two directions and illuminates the sample with interference fringes of the excitation light, a control unit that controls the direction and phase of the interference fringes, An imaging optical system that forms an image of a sample irradiated with fringes, an imaging element that captures an image formed by the imaging optical system to generate a first image, and a plurality of first images generated by the imaging element And a demodulation unit for generating a second image by using the structure illumination microscope.

  According to the second aspect of the present invention, irradiating the sample with activation light for activating the fluorescent substance contained in the sample from a first direction, and activating light from a second direction different from the first direction, Irradiating the sample with the excitation light for exciting the fluorescent substance with interference fringes, controlling the direction and phase of the interference fringes, forming an image of the sample irradiated with the interference fringes, An observation method is provided, which includes imaging to generate a first image, and generating a second image using a plurality of first images generated by the imaging.

  According to a third aspect of the present invention, the computer irradiates the sample with the activation light for activating the fluorescent substance contained in the sample from the first direction, and irradiates the computer with the second light different from the first direction. Irradiating the sample with excitation light for exciting the fluorescent substance from the direction with interference fringes, controlling the direction and phase of the interference fringes, and forming an image of the sample irradiated with the interference fringes. And generating a first image by capturing an image, and generating a second image by using a plurality of first images generated by the capturing. Is done.

  According to the present invention, it is possible to provide a structured illumination microscope, an observation method, and a control program capable of generating a super-resolution image for a sample having a thickness exceeding the depth of focus of the imaging optical system.

It is a figure showing the structured microscope concerning a 1st embodiment. It is a figure showing an example of a mask. FIG. 6 is a diagram illustrating a polarization state of diffracted light in a second illumination optical system. FIG. 3 is a diagram illustrating a position and a polarization state of a light-converging region on a pupil plane. It is a figure which shows the illuminating area | region of the activation light from the activation illumination part, and the excitation illumination part. 5 is a flowchart illustrating an example of an operation of the structured illumination microscope. It is a flowchart which shows the process which detects the activation level of a fluorescent dye. It is a flowchart which shows the process which detects the inactivation level of a fluorescent dye. It is a figure showing the 1st modification of an activation lighting part. It is a figure showing the 2nd modification of an activation lighting part. It is a figure showing the 3rd modification of an activation lighting part. It is a figure showing the 4th modification of an activation lighting part. It is a figure showing the 5th modification of an activation lighting part. It is a figure showing the 6th modification of an activation lighting part. It is a figure showing the 7th modification of an activation lighting part. It is a figure showing the 8th modification of an activation lighting part. It is a figure showing the 9th modification of an activation lighting part. It is a figure showing the 10th modification of an activation lighting part. It is a figure showing the 11th modification of an activation lighting part. It is a figure which shows the modification of a deactivation illumination part. It is a figure showing the structured microscope concerning a 2nd embodiment.

[First Embodiment]
FIG. 1 is a diagram showing a structured illumination microscope 1 (observation system) according to the present embodiment. The structured illumination microscope 1 is used for observing a sample S stained with a fluorescent dye (fluorescent substance). The sample S includes, for example, fluorescent cells (cells stained with a fluorescent dye) arranged on a parallel plate-like glass surface and fluorescent living cells (moving cells stained with a fluorescent dye) existing in a petri dish. ).

  The fluorescent dye contained in the sample S is activated upon receiving the activation light, and emits fluorescence upon receiving the excitation light in the activated state. When the fluorescent dye receives an inactivating light in an activated state, it emits fluorescence to be in an inactivated state. The wavelength of the deactivating light may be the same as the wavelength of the excitation light, for example. Such fluorescent dyes include, for example, dye pairs in which two types of cyanine dyes are bonded (eg, Cy3-Cy5 dye pairs, Cy2-Cy5 dye pairs, Cy3-Alexa647 dye pairs, and the like). For example, in the Cy3-Cy5 dye pair, Cy3 is a site that activates Cy5, and Cy5 is activated by irradiating green light of 532 nm. When red light of 640 nm corresponding to the absorption wavelength of Cy5 is irradiated, Cy5 emits fluorescence and is inactivated.

  The structured illumination microscope 1 has a 2D-SIM mode for generating a two-dimensional super-resolution image of a part of the sample S in the thickness direction. The structured illumination microscope 1 includes a stage 2, an activation illumination unit 3, an excitation illumination unit 4, an imaging unit 5, a demodulation unit 6, and a control unit 7. The structured illumination microscope 1 operates roughly as follows. The stage 2 holds the sample S. The activation illumination unit 3 irradiates the sample S with activation light in the form of a sheet. The activation illumination unit 3 selectively activates a part of the sample S in the thickness direction, and maintains another part of the sample S in an inactive state. The excitation illumination section 4 forms interference fringes with the excitation light. The excitation illumination unit 4 illuminates the activated portion of the sample S with interference fringes from a direction perpendicular to the light irradiation direction of the activation illumination unit 3. The imaging unit 5 captures a modulated image of the sample S modulated by the interference fringes. The demodulation unit 6 demodulates the modulated image captured by the imaging unit 5 and generates an image (super-resolution image) of the sample S. The control unit 7 controls each unit of the structured illumination microscope 1 to execute various processes.

  Next, each part of the structured illumination microscope 1 will be described. In the following description, the first direction (light irradiation direction) in which the activation illumination unit 3 irradiates the sample with light is defined as the X direction. Further, a second direction (light irradiation direction) in which the excitation illumination unit 4 irradiates the sample S with light is defined as a Z direction. A third direction perpendicular to each of the X direction and the Z direction is defined as a Y direction. The X direction and the Y direction are set, for example, in the horizontal direction, and the Z direction is set, for example, in the vertical direction. In addition, + or-is added to each direction to indicate the direction and the direction as appropriate.

  The stage 2 can mount the sample S on its upper surface. The stage 2 can move on the upper surface in the Z direction with the sample S placed on the upper surface. The stage 2 is controlled by the control unit 7 and can adjust the position of the upper surface in the Z direction. The control unit 7 can control the position (height) of the sample S disposed on the upper surface in the Z direction by controlling the stage 2. The stage 2 may move the sample S in at least one of the X direction and the Y direction, or may not move the sample S in the Z direction.

  The activation illumination unit 3 includes a light source unit 10 and a first illumination optical system 11. The light source unit 10 is controlled by the control unit 7 and emits activation light. The activating light includes light of a first wavelength band that activates a predetermined fluorescent substance (fluorescent dye). The light source unit 10 includes, for example, a laser diode (LD), but may include a light emitting diode (LED). The light source unit 10 may emit either coherent light or incoherent light as the activating light. The light source unit 10 may emit any of linearly polarized light, elliptically polarized light, and non-polarized light as the activating light. Note that the structured illumination microscope 1 does not need to include the light source unit 10. For example, the light source unit 10 may be provided exchangeably (removably) with the structured illumination microscope 1, and may be an external device of the structured illumination microscope 1.

  The first illumination optical system 11 guides the activation light from the light source unit 10 to the sample S. In the present embodiment, the optical axis AX1 of the first illumination optical system 11 is set so that the light irradiation direction of the activation illumination unit 3 is in the X direction. The first illumination optical system 11 includes a condenser lens 12, an optical fiber 13, a collimator lens 14, a cylindrical lens 15, a slit 16, and a lens group 17 on the light emission side of the light source unit 10.

  The condensing lens 12 condenses the activating light from the light source unit 10 so as to fit on the incident end face of the optical fiber 13. The optical fiber 13 is a light guide member that guides light incident on the incident end face to the output end face. The collimator lens 14 collimates the activation light emitted from the emission end face of the optical fiber 13. The cylindrical lens 15 is an optical member whose refractive power (power) in the XZ plane is stronger than that in the XY plane. The collimator lens 14 and the cylindrical lens 15 shape the cross-sectional shape of the activation light in the sample S (the illumination area of the activation illumination unit 3) into a shape in which the Z direction is the shorter direction among the Y direction and the Z direction.

  The slit 16 is a field stop (a stop member), and is arranged at a position optically conjugate with the illumination area of the activation illumination unit 3. The slit 16 has an opening through which at least a part of the activating light from the light source unit 10 passes. The shape of the opening is such that the dimension in the Z direction is shorter than the dimension in the Y direction, and is, for example, a rectangular shape having short sides parallel to the Z direction. The aperture ratio of the slit 16 may be a fixed value or a variable value. For example, at least one of the shape and size of the opening of the slit 16 may be variable. For example, the opening of the slit 16 may have a variable inner size in at least one of the Y direction and the Z direction.

  The lens group 17 is, for example, a projection optical system, and projects an image of the opening of the slit 16 onto the illumination area of the activation illumination unit 3. The activating light passing through the opening of the slit 16 is applied to the sample S via the lens group 17. By reducing the numerical aperture (NA) on the light exit side (eg, lens group 17) of the first illumination optical system 11, the spread angle of the activating light when exiting from the first illumination optical system 11 is reduced. can do. As a result, a change in the thickness of the activation light (the width of the optical path in the Z direction) of the sample S can be reduced.

  As described above, the activation illumination unit 3 can irradiate a part of the sample S in the Z direction with the activation light and selectively activate the sheet-shaped part of the sample S. The activated sheet-like portion is referred to as an activation layer in the following description.

  In the present embodiment, the structured illumination microscope 1 includes a passivation illumination unit 20. The deactivating illumination unit 20 irradiates the sample S with deactivating light for deactivating the fluorescent dye activated by the activating light. The deactivating illumination unit 20 includes a light source unit 21, a mirror 22, a dichroic mirror 23, and the first illumination optical system 11. The light source unit 21 emits inactivating light including light in the second wavelength band that causes a predetermined fluorescent dye to transition from an activated state to an inactive state.

  The deactivating light emitted from the light source unit 21 is reflected by the mirror 22 and enters the dichroic mirror 23. The mirror 22 and the dichroic mirror 23 are light guides that guide the inactivating light emitted from the light source 21 to the first illumination optical system 11. The dichroic mirror 23 is disposed on an optical path between the light source unit 10 and the condenser lens 12. The dichroic mirror 23 has a wavelength selection film inside. The wavelength selection film has a characteristic that the activating light from the light source unit 10 is transmitted and the inactivating light from the light source unit 21 is reflected. The deactivating light that has entered the dichroic mirror 23 is reflected by the wavelength selection film, and enters the first illumination optical system 11 through substantially the same optical path as the activating light from the light source unit 10. The deactivating light that has entered the first illumination optical system 11 is formed into a sheet shape and irradiated onto the sample S, similarly to the activating light. Note that the structured illumination microscope 1 does not need to include the light source unit 21. For example, the light source unit 21 may be exchangeably (removably) provided to the structured illumination microscope 1 and may be an external device of the structured illumination microscope 1.

  In the present embodiment, the range in which the inactivating illumination unit 20 irradiates the inactivating light (hereinafter, referred to as an illumination area of the inactivating illumination unit 20) corresponds to the range in which the activation illumination unit 3 irradiates the activating light ( Hereinafter, the illumination area of the activation illumination section 3). The illumination area of the inactivation illumination unit 20 may be set to be wider than the illumination area of the activation illumination unit 3. For example, when the opening of the slit 16 of the first illumination optical system 11 is variable, the opening width (for example, the inner diameter in the Z direction) of the slit 16 is determined when the inactivation light passes through the first illumination optical system 11. , May be set to be larger than when the activating light passes through the first illumination optical system 11. In this case, it is possible to suppress a portion that is not inactivated from remaining in the activated portion in the sample S. Note that the control unit 7 may control a driving unit (not shown) that drives the opening of the slit 16 to control the aperture ratio of the slit 16.

  The excitation illumination section 4 illuminates the sample with interference fringes formed by the excitation light. The excitation illumination unit 4 includes a light source unit 30 and a second illumination optical system 31. The light source unit 30 is controlled by the control unit 7 and emits excitation light. The excitation light includes light in a wavelength band that excites a predetermined fluorescent dye, and includes, for example, light having the same wavelength as the inactivation light. The light source unit 30 includes, for example, a laser diode (LD) and may emit coherent light as excitation light, or may include a light emitting diode (LED) and emit incoherent light as excitation light. Here, the light source unit 30 emits linearly polarized light as activation light. Note that the structured illumination microscope 1 does not need to include the light source unit 30. For example, the light source unit 30 may be exchangeably (removably) provided to the structured illumination microscope 1 and may be a device external to the structured illumination microscope 1.

  The second illumination optical system 31 irradiates the sample S with excitation light via the objective lens 32 and illuminates the sample S with incident light. The optical axis of the objective lens 32 (the optical axis on the light emission side of the second illumination optical system 31) is set in the Z direction. The optical axis AX2 of the second illumination optical system 31 is bent by the dichroic mirror 33 on the light source unit 30 side of the objective lens 32. The optical axis AX2 of the second illumination optical system 31 is arbitrarily set in the optical path closer to the light source unit 30 than the dichroic mirror 33, and is set, for example, in a direction parallel to the XY plane.

  The second illumination optical system 31 includes a condenser lens 35, an optical fiber 36, and a collector lens 37 on the light emission side of the light source unit 30. The condenser lens 35 condenses the excitation light from the light source unit 30 so as to fit on the incident end face of the optical fiber 36. The optical fiber 36 is a light guide member that guides light incident on the incident end face to the output end face. The optical fiber 36 is, for example, a polarization-maintaining single-mode fiber, and guides the pump light without substantially changing the polarization state of the pump light. The excitation light emitted from the optical fiber 36 is, for example, linearly polarized light in the Z direction. The collector lens 37 collimates the excitation light emitted from the emission end face of the optical fiber 36.

  The second illumination optical system 31 includes a polarizing plate 38, a diffraction grating 39, a half-wave plate 40, and a lens 41 on the light emission side of the collector lens 37. In the present embodiment, the optical axis AX2 of the second illumination optical system 31 is set parallel to the X direction in the optical path from the collector lens 37 to the dichroic mirror 33. The polarizing plate 38 transmits linearly polarized light parallel to the transmission axis and blocks (shields) linearly polarized light perpendicular to the transmission axis. The polarizing plate 38 has, for example, a transmission axis parallel to the Z direction, and blocks components of the excitation light whose polarization state is disturbed, stray light from the light source unit 30 side of the polarizing plate 38, and the like.

  The diffraction grating 39 is a branching unit that branches the excitation light from the light source unit 30. The diffraction grating 39 splits the excitation light into a plurality of light beams by diffraction. In the present embodiment, the second illumination optical system 31 generates interference fringes in the sample by two-beam interference of + 1st-order diffracted light and −1st-order diffracted light generated by the diffraction grating 39. In FIG. 1, the 0th-order diffracted light is indicated by a solid line, the + 1st-order diffracted light is indicated by a chain line, and the -1st-order diffracted light is indicated by a two-dot chain line.

  The diffraction grating 39 has, for example, a one-dimensional periodic structure. This periodic structure may be a structure in which the density (transmittance) changes periodically, or a structure in which the step (phase difference) changes periodically. When the diffraction grating 39 is of a phase type, the diffraction efficiency of ± first-order diffracted light is high, and light loss can be reduced when ± first-order diffracted light is used for forming interference fringes. Note that the period of the interference fringes formed on the sample S may be a period slightly smaller than the resolution limit of the imaging optical system 55 (described later) of the imaging unit 5. By setting the spatial modulation frequency of the interference fringes as close as possible to the limit that can be formed by the imaging optical system 55, the resolution of the sample image obtained by the demodulation processing can be further increased.

  By the way, when an image of one sample is acquired by the 2D-SIM, for example, nine modulation images are used for the operation (demodulation operation) in the demodulation process. Therefore, in the 2D-SIM, the direction of the interference fringe is changed in three ways, and the phase of the interference fringe is changed in three ways for each direction of the interference fringe, and the combination of the direction and the phase of the interference fringe is changed. Modulated images are picked up in nine different states.

  The structured illumination microscope 1 according to the present embodiment uses nine modulated images for demodulation calculation when acquiring an image of the activation layer of the sample S (described later). The direction of the interference fringes corresponds to the direction in which the light intensity changes periodically in the interference fringes (the direction orthogonal to the direction in which the light intensity hardly changes). The direction of the interference fringes can be changed, for example, by rotating (rotating) the diffraction grating 39 around the optical axis AX2 of the second illumination optical system 31. The phase of the interference fringe corresponds to the position of a bright part and the position of a dark part in the interference fringe. The phase of the interference fringes can be changed, for example, by moving the diffraction grating 39 in a direction crossing the optical axis AX2 of the second illumination optical system 31.

  In the present embodiment, the second illumination optical system 31 includes a rotation drive unit 42 that rotates the diffraction grating 39 around the optical axis AX2. The rotation drive unit 42 includes, for example, an actuator such as an electric motor. The rotation drive unit 42 is controlled by the control unit 7 and rotates the diffraction grating 39. The control unit 7 switches the rotation position of the diffraction grating 39 between the first rotation position, the second rotation position, and the third rotation position by controlling the rotation driving unit 42 (shown later in FIG. 3). . For example, the second rotation position is a rotation position rotated by 120 degrees from the first rotation position, and the third rotation position is rotated by 120 degrees from the second rotation position (240 degrees from the first rotation position). This is the rotated position.

  In the present embodiment, the second illumination optical system 31 includes a parallel drive unit 43 that drives the diffraction grating 39 in a direction perpendicular to the optical axis AX2. The parallel driving unit 43 includes an actuator such as a piezo element. The parallel drive unit 43 is controlled by the control unit 7 and moves the diffraction grating 39 in parallel. The control unit 7 controls the position of the diffraction grating 39 in a direction parallel to the YZ plane by controlling the parallel driving unit 43. The control unit 7 controls the parallel driving unit 43 such that, for example, every time the position of the diffraction grating 39 is changed, the phase of the interference fringe changes by a predetermined amount. This predetermined amount is set to, for example, about 1/3 of the period of the interference fringes (eg, 2π / 3 phase).

  The half-wave plate 40 adjusts the polarization state of the excitation light from the diffraction grating 39 so that the polarization state of the excitation light when entering the sample S is S-polarized (described later in FIGS. 3 and 4). ). The lens 41 focuses the excitation light from the half-wave plate 40 on the pupil conjugate plane 44. The excitation light is collected by the lens 41 at a position corresponding to the order of the diffracted light. For example, the 0th-order diffracted light of the excitation light is focused toward the optical axis AX2 on the pupil conjugate plane 44. The + 1st-order diffracted light of the excitation light is collected toward a position on the pupil conjugate plane 44 shifted from the optical axis AX2. Of the excitation light, the -1st-order diffracted light is converged on the optical axis AX2 on the pupil conjugate plane 44 toward a position symmetrical with the converging position of the + 1st-order diffracted light.

  The second illumination optical system 31 includes a mask 45, a lens 46, a field stop 47, a lens 48, an excitation filter 49, a dichroic mirror 33, and an objective lens 32 on the light emission side of the lens 41.

  The mask 45 (light flux selecting unit) passes, among the plurality of diffracted lights from the diffraction grating 39, the diffracted light of the order used for forming the interference fringes, and blocks the diffracted light of the order not used for the formation of the interference fringes. . In the present embodiment, the diffracted light used to form the interference fringes is ± 1st order, and the mask 45 transmits the ± 1st order diffracted light and blocks the 0th order diffracted light and other diffracted light. The mask 45 is arranged, for example, on the pupil conjugate plane 44 (position of the aperture stop). The mask 45 only needs to be arranged at a position where none of the optical path of the 0th-order diffracted light, the optical path of the + 1st-order diffracted light, and the optical path of the -1st-order diffracted light overlap. Good.

  FIG. 2A is a diagram illustrating an example of the mask 45. A portion of the mask 45 including the optical axis AX2 is a portion where the zero-order diffracted light is incident, and is a light shielding portion. The mask 45 has a plurality of openings 45a to 45f arranged apart from the optical axis AX2 of the second illumination optical system 31. The openings 45a to 45d are arranged every 60 degrees around the optical axis AX2. The openings 45a and 45d are respectively arranged at positions where the + 1st-order diffracted light L1 and the -1st-order diffracted light L2 pass when the diffraction grating 39 is arranged at the first rotation position. The opening 45c and the opening 45f are arranged at positions where the + 1st-order and -1st-order diffracted lights pass when the diffraction grating 39 is arranged at the second rotation position, respectively. The opening 45b and the opening 45e are arranged at positions where the + 1st-order and -1st-order diffracted lights pass when the diffraction grating 39 is arranged at the third rotation position, respectively. The inner dimensions of the openings 45a to 45f are set so as to secure an effective diameter through which a light beam used for forming interference fringes passes.

  FIG. 2B is a diagram illustrating another example of the mask 45. In the mask 45, the optical axis AX2 and its periphery are disk-shaped light shielding portions 50. The mask 45 has an annular outer edge portion 51, and the light-shielding portion 50 is supported by the outer edge portion 51 by a band-shaped support portion 52. The support portion 52 is arranged at a position where ± 1st-order diffracted light does not pass even when the diffraction grating 39 is arranged at any of the first to third rotational positions.

  The lens 46 condenses the excitation light (± first-order diffracted light) from the mask 45 at the same position for each diffracted light generated at each position of the diffraction grating 39. The lens 41 and the lens 46 form an image of the diffraction grating 39. The field stop 47 is arranged at a position optically conjugate with the diffraction grating 39. The field stop 47 has an opening through which excitation light passes, and defines an illumination area of the second illumination optical system 31 by the shape and size of the opening.

  The excitation filter 49 blocks at least a part of the light from the lens 46 other than the excitation wavelength. For example, the excitation filter 49 cuts off stray light from the light source unit 30 side than the excitation filter 49. The dichroic mirror 33 has a characteristic of reflecting the excitation light from the light source unit 30 and transmitting the fluorescence from the sample S. The dichroic mirror 33 is provided, for example, at an angle of about 45 ° with respect to the X direction. The excitation light from the excitation filter 49 is reflected by the dichroic mirror 33, the optical path is bent by about 90 °, travels in the + Z direction, and enters the objective lens 32. The objective lens 32 forms an image of the diffraction grating 39 at a position optically conjugate with the field stop 47.

  Here, the optical path and the polarization state of the excitation light depending on the rotational position of the diffraction grating 39 will be described. FIG. 3 is a diagram illustrating an optical path and a polarization state of the excitation light at each rotation position of the diffraction grating 39. FIG. 4 is a diagram showing the condensing region on the pupil plane 32a of the objective lens 32 and the polarization state of the excitation light at each rotational position of the diffraction grating 39. In FIGS. 3 and 4, the arrows in the circles indicate the polarization state of the excitation light.

  A symbol Q1 indicates a state where the diffraction grating 39 is arranged at the first rotation position. The first rotation position is arbitrarily set, for example, a rotation position where the periodic direction D1 of the diffraction grating 39 is parallel to the Z direction. The transmission axis 38a of the polarizing plate 38 is set, for example, parallel to the Z direction, and the polarization state Q1a of the excitation light passing through the polarizing plate 38 is linearly polarized light in the Z direction. The + 1st-order diffracted light L1 and the -1st-order diffracted light L2 generated by the diffraction grating 39 are branched away from each other in the periodic direction D1 of the diffraction grating 39. In the optical path between the diffraction grating 39 and the half-wave plate 40, the polarization state Q1b of each diffracted light is, for example, linearly polarized light in the Z direction. In the state Q1, the fast axis 40a of the half-wave plate 40 is set, for example, in a direction forming an angle of −45 ° with the Y direction. Of the diffracted lights that have passed through the half-wave plate 40, the 0th-order diffracted light is blocked by the mask 45 and is excluded from the optical path toward the sample S. The polarization states Q1c of the + 1st-order diffracted light L1 and the −1st-order diffracted light L2 that have passed through the mask 45 are linearly polarized light in a direction (Y direction) orthogonal to the periodic direction D1 of the diffraction grating 39, respectively.

  As shown in the state Q1 of FIG. 4, the + 1st-order diffracted light L1 and the -1st-order diffracted light L2 are respectively condensed on the condensing area on the pupil plane 32a of the objective lens 32. Here, the rotational position θ about the optical axis AX2 is represented by an angle where the rotational position in the radial direction parallel to the X direction is 0 ° and the clockwise direction when viewed from the −Z direction is positive. The condensing area of the + 1st-order diffracted light L1 is arranged at a rotation position of θ = 0 °, and the condensing area of the −1st-order diffracted light L2 is arranged at a rotation position of θ = 180 °. The direction D4 connecting the converging area of the + 1st-order diffracted light L1 and the converging area of the -1st-order diffracted light L2 is parallel to the X direction, and the plane of incidence of the ± 1st-order diffracted light on the sample S includes the optical axis AX2. This is a plane (XZ plane) parallel to D4. The ± first-order diffracted light is linearly polarized light in the Y direction, and is S-polarized light with respect to the sample S.

  3 and 4, reference numeral Q2 indicates a state where the diffraction grating 39 is arranged at the second rotation position. In the state Q2, the angle between the periodic direction D2 of the diffraction grating 39 and the Z direction is, for example, 120 °. The polarization state Q2a of the excitation light passing through the polarizing plate 38 is, for example, linearly polarized light in the Z direction. The + 1st-order diffracted light L1 and the -1st-order diffracted light L2 generated by the diffraction grating 39 are branched away from each other in the periodic direction D2 of the diffraction grating 39. In the optical path between the diffraction grating 39 and the half-wave plate 40, the polarization state Q2b of each diffracted light is, for example, linearly polarized light in the Z direction. In the state Q2, the fast axis 40a of the half-wave plate 40 is set, for example, in a direction forming an angle of 15 ° with the Y direction. Of the diffracted lights that have passed through the half-wave plate 40, the 0th-order diffracted light is blocked by the mask 45 and is excluded from the optical path toward the sample S. The polarization states Q2c of the + 1st-order diffracted light L1 and the -1st-order diffracted light L2 that have passed through the mask 45 are linearly polarized light in a direction orthogonal to the periodic direction D2 of the diffraction grating 39, respectively.

  As shown in a state Q2 in FIG. 4, the + 1st-order diffracted light L1 and the −1st-order diffracted light L2 are respectively condensed on a condensing area on the pupil plane 32a of the objective lens 32. The converging region of the + 1st-order diffracted light L1 is arranged at a rotational position of θ = 120 °, and the converging region of the −1st-order diffracted light L2 is arranged at a rotational position of θ = 300 °. The direction D5 connecting the converging region of the + 1st-order diffracted light L1 and the converging region of the -1st-order diffracted light L2 is a direction having an angle of 120 ° with respect to the + X direction as positive clockwise. The plane of incidence of the ± 1st-order diffracted light on the sample S is a plane that includes the optical axis AX2 and is parallel to the direction D5. The ± first-order diffracted light is linearly polarized light in a direction orthogonal to the direction D5, and is S-polarized light with respect to the sample S.

  Further, in FIGS. 3 and 4, reference numeral Q3 indicates a state where the diffraction grating 39 is arranged at the third rotation position. In the state Q3, the angle between the periodic direction D3 of the diffraction grating 39 and the Z direction is, for example, 240 °. The polarization state Q3a of the excitation light that has passed through the polarizing plate 38 is, for example, linearly polarized light in the Z direction. The + 1st-order diffracted light L1 and the -1st-order diffracted light L2 generated by the diffraction grating 39 are branched away from each other in the periodic direction D3 of the diffraction grating 39. In the optical path between the diffraction grating 39 and the half-wave plate 40, the polarization state Q3b of each diffracted light is, for example, linearly polarized light in the Z direction. In the state Q3, the fast axis 40a of the half-wave plate 40 is set, for example, in a direction forming an angle of 75 ° with the Y direction. Of the diffracted lights that have passed through the half-wave plate 40, the 0th-order diffracted light is blocked by the mask 45 and is excluded from the optical path toward the sample S. The polarization states Q3c of the + 1st-order diffracted light L1 and the −1st-order diffracted light L2 that have passed through the mask 45 are linearly polarized light in a direction orthogonal to the periodic direction D3 of the diffraction grating 39, respectively.

  As shown in the state Q3 of FIG. 4, the + 1st-order diffracted light L1 and the -1st-order diffracted light L2 are respectively condensed on the condensing area on the pupil plane 32a of the objective lens 32. The converging region of the + 1st-order diffracted light L1 is arranged at a rotational position of θ = 240 °, and the converging region of the −1st-order diffracted light L2 is arranged at a rotational position of θ = 60 °. The direction D6 connecting the converging region of the + 1st-order diffracted light L1 and the converging region of the -1st-order diffracted light L2 is a direction at an angle of 240 ° from the + X direction with the clockwise direction being positive. The plane of incidence of the ± 1st-order diffracted light on the sample S is a plane that includes the optical axis AX2 and is parallel to the direction D6. The ± first-order diffracted light is linearly polarized light in a direction orthogonal to the direction D6, and is S-polarized light with respect to the sample S.

  In order to adjust the polarization state of the ± 1st-order diffracted light according to the rotational position of the diffraction grating 39, the half-wave plate 40 may be rotated. For example, when the control unit 7 controls the rotation driving unit 42 to rotate the diffraction grating 39 by 120 °, the control unit 7 may rotate the half-wave plate 40 by 60 °. In the mask 45 shown in FIG. 2A, the half-wave plate 40 may be fixed to each opening through which ± 1st-order diffracted light passes according to the rotational position of the diffraction grating 39. In this case, the direction of the fast axis of the half-wave plate 40 may be set for each opening according to the position of the opening.

  As described above, the excitation illumination unit 4 shown in FIG. 1 forms interference fringes by ± 1st-order diffracted light in the illumination area set in the activation layer of the sample S, The converted fluorescent substance emits fluorescence by the excitation light. With this fluorescence, a modulated image in which the fluorescence density distribution of the sample S is modulated by interference fringes is obtained.

  The imaging unit 5 captures a modulated image of the sample S modulated by interference fringes. The imaging unit 5 includes an imaging optical system 55 and an imaging device 56. The imaging optical system 55 includes an objective lens 32, a dichroic mirror 33, a barrier filter 57, and a lens 58.

  The fluorescence from the sample S passes through the objective lens 32, is converted into parallel light, passes through the dichroic mirror 33, and enters the barrier filter 57. The barrier filter 57 blocks at least a part of light in a wavelength band other than the wavelength of the fluorescence from the sample S. The lens 58 forms an image plane with the sample S (the illumination area of the excitation illumination unit 4) as an object plane together with the objective lens 32. That is, the objective lens 32 and the lens 58 form a modulated image of the sample S. The focal position of the imaging optical system 55 is set, for example, in the illumination area of the activation illumination unit 3 in the Z direction.

  The imaging element 56 includes, for example, an image sensor such as a CCD sensor or a CMOS sensor. The image sensor 56 has a light receiving surface on which a plurality of pixels are arranged. The light receiving surface is arranged on the image plane of the imaging optical system 55 (a plane optically conjugate with the sample S). On the light receiving surface, a photoelectric conversion unit such as a photodiode is disposed in each pixel, and the imaging element 56 converts the fluorescence from the sample S into electric charges by the photoelectric conversion unit and detects the fluorescence from the sample S.

  In this way, the imaging unit 5 captures the modulated image and supplies the captured result (hereinafter, referred to as a modulated image) to the demodulation unit 6. The demodulation unit 6 performs a demodulation process using the imaging result of the modulated image, and generates an image of the sample S. The demodulation unit 6 can generate a restored image using a plurality of modulated images captured in a state where the directions and directions of the interference fringes are different from each other. The demodulation unit 6 may perform demodulation processing by a method disclosed in, for example, US Pat. No. 8,115,806, but is not limited to this method. For example, it may be performed by the method disclosed in U.S. Pat. No. 8,115,806.

  In the present embodiment, the structured illumination microscope 1 holds a portion to be observed in the thickness direction of the sample S (hereinafter, referred to as an observation target surface) in an illumination area of the activation illumination unit 3, and the activation illumination unit 3 The observation target surface can be selectively activated. Further, the structured illumination microscope 1 can irradiate interference fringes on the observation target surface by the excitation illumination unit 4 while keeping at least a part of the thickness portion of the sample S other than the observation target surface in an inactive state. In this manner, the structured illumination microscope 1 can selectively generate fluorescence from the observation target surface of the sample S, and can capture a modulated image of the observation target surface by the imaging unit 5. Then, by demodulating this modulated image, a super-resolution image exceeding the resolution limit of the imaging optical system 55 is obtained.

  The structured illumination microscope 1 according to the present embodiment can suppress the generation of fluorescence in a portion other than the observation target surface of the sample S, and thus can reduce the background light relative to the fluorescence from the observation target surface. As a result, when the structured illumination microscope 1 generates a super-resolution image for the sample S having a thickness exceeding the depth of focus of the imaging optical system 55, it is possible to suppress a decrease in image quality.

  FIG. 5 is a diagram illustrating the activation light L4 from the activation illumination unit 3 and the illumination region IR of the excitation illumination unit 4. FIG. 5 shows a state where the optical axis AX1 of the activation illumination unit 3 is set to pass through the optical axis AX2 of the excitation illumination unit 4. In the present embodiment, the optical axis AX1 of the activation illumination unit 3 is fixed so as to pass through the optical axis AX2 of the excitation illumination unit 4.

  The illumination area IR of the excitation illumination section 4 is set, for example, at a position optically conjugate with the light receiving surface of the image sensor 56. The range in which the activation illumination unit 3 emits the activation light L4 is set to, for example, a range including the illumination region IR. For example, the activation illumination unit 3 irradiates the sheet-like activation light L4 over a wider range than the illumination region IR on a plane parallel to the XY plane.

  The range in which the activation illumination unit 3 emits the activation light L4 may be set at a position shifted from the illumination region IR in the Z direction. In this case, after activating a part of the sample S in the Z direction, the sample S is moved in the Z direction by the stage 2, and the activation layer is arranged in the illumination region IR of the excitation illumination unit 4 and is moved to the activation layer. Irradiation of the excitation light and imaging of the modulation image of the activation layer may be executed. In the structured illumination microscope 1, the relative position between the range in which the activation illumination unit 3 irradiates the activation light and the illumination region IR of the excitation illumination unit 4 may be variable (described in a modified example). .

  The thickness B of the activation light L4 in the illumination region IR of the excitation illumination section 4 is set to, for example, 1 μm or less, and is set to be equal to or less than the thickness of the sample S in the Z direction. By changing the position (height) of the activation light L4 in the Z direction of the stage 2, it is possible to acquire the respective modulated images of the plurality of observation target surfaces of the sample S at different positions in the Z direction. When performing observation while changing the position of the stage 2 in the Z direction in a stepwise manner, the step amount of the position of the stage 2 in the Z direction may be set to, for example, the same value as the thickness B of the activation light, The value may be set to a value larger than the thickness B or may be set to a value smaller than the thickness B. Further, the thickness B of the activation light L4 may be set, for example, in a range equal to or less than the depth of focus of the imaging optical system 55. In this case, it is difficult to detect fluorescence from a portion other than the observation target surface in the sample S.

  In the present embodiment, the structured illumination microscope 1 includes a control device CONT. The control unit 7 and the demodulation unit 6 are provided in the control device CONT. The control device CONT includes, for example, a computer system, and executes various processes according to a control program read from a storage device (not shown). The control program includes, for example, irradiating a computer with activation light for activating a fluorescent substance contained in a sample from a first direction, and irradiating the computer with fluorescent light from a second direction different from the first direction. Irradiating the sample with excitation light to excite the substance using interference fringes, controlling the direction and phase of the interference fringes, forming an image of the sample irradiated with the interference fringes, and capturing the image Then, control of processing including generating a first image and generating a second image using a plurality of first images generated by imaging is performed.

  The control device CONT is connected to a display device 59 such as a liquid crystal display, and causes the display device 59 to display an image indicating an observation result (eg, a super-resolution image), an image indicating settings of the structured illumination microscope 1, and the like. Can be. An input device (not shown) such as a keyboard, a mouse, and a touch panel is connected to the control device CONT. This input device accepts, for example, input of information such as observation conditions, settings of the structured illumination microscope 1, and instructions from a user. The observation conditions include, for example, the position of the observation target portion of the sample S. The setting of the structured illumination microscope 1 includes, for example, setting of an observation mode. The command includes, for example, starting, suspending, resuming, and ending the observation.

  Next, an observation method of the sample S will be described based on the operation of the structured illumination microscope 1 as described above. FIG. 6 is a flowchart showing the operation of the structured illumination microscope 1. The structured illumination microscope 1 sets the height (position in the Z direction) of the sample S in step S1. For example, the control unit 7 of the control device CONT arranges the observation target surface of the sample S specified by the user in the illumination area of the activation illumination unit 3 by controlling the stage 2.

  In step S2, the structured illumination microscope 1 irradiates the sample S with activation light from the activation illumination unit 3. For example, the control unit 7 controls the light source unit 10 to irradiate the sample S with a predetermined amount of activation light. The set value of the light amount is defined, for example, by the irradiation time, the light emission intensity of the light source unit 10, the power supplied to the light source unit 10, and the like. The set value of the light amount is set to, for example, a level at which the activation of the fluorescent dye on the observation target surface of the sample S can be saturated. The structured illumination microscope 1 stops the irradiation of the activation light from the activation illumination unit 3 to the sample S, for example, by the control unit 7 controlling the light source unit 10 in step S3.

  In step S4, the structured illumination microscope 1 sets conditions for the interference fringes formed by the excitation illumination unit 4. The condition of the interference fringes includes at least one of the phase and the direction of the interference fringes. For example, the controller 7 controls the position of the diffraction grating 39 by controlling the parallel drive unit 43 when changing the phase of the interference fringe in the next imaging. When changing the direction of the interference fringes in the next imaging, the control unit 7 controls the rotation driving unit 42 to control the rotation position of the diffraction grating 39. The processing in step S4 may be performed in parallel with the processing in step S2, or may be performed in a period that does not overlap with the processing in step S2.

  After the processing in step S3 and the processing in step S4 are completed, the structured illumination microscope 1 irradiates the sample S with excitation light from the excitation illumination unit 4 in step S5. In addition, the structured illumination microscope 1 images the sample S irradiated with the excitation light by the imaging unit 5 in step S6. For example, the control unit 7 controls the light source unit 30 to irradiate the excitation illumination unit 4 with the excitation light, and causes the imaging unit 5 to image the sample S at a predetermined timing with respect to the excitation light irradiation.

  In step S7, the structured illumination microscope 1 determines whether or not a predetermined number of images have been captured. For example, when the number of modulated images required for the demodulation operation by the demodulation unit 6 is nine, the control unit 7 determines whether or not these nine modulated images have been imaged. When the control unit 7 determines that the predetermined number of imagings has not been completed (Step S7; No), the control unit 7 executes the processing from Step S2 to Step S6. When determining that the predetermined number of images have been captured (Step S7; Yes), the control unit 7 causes the demodulation unit 6 to execute demodulation processing based on the acquired modulated image (Step S8).

  In step S9, the control unit 7 determines whether or not imaging has been completed in the observation region in the depth direction of the sample S. The observation area in the depth direction is defined, for example, by observation conditions specified by the user. For example, the user can specify an observation region by specifying two positions, an observation start position and an observation end position, in the depth direction. In the present embodiment, the control unit 7 of the control device CONT moves the stage 2 until the observation end position specified by the user is reached, changes the observation target surface, and repeats the process of acquiring the modulated image, thereby obtaining a predetermined image. A super-resolution image of the thickness portion is generated. When the control unit 7 determines that the imaging of the observation region in the predetermined depth direction has not been completed (Step S9; No), the control unit 7 inactivates the sample S from the inactivation illumination unit 20 in Step S10. Irradiate light. The control unit 7 stops the irradiation of the deactivating light by the deactivating illumination unit 20, for example, after the deactivating illumination unit 20 emits a predetermined amount of deactivating light (step S11). ). After the processing of step S11 is completed, the structured illumination microscope 1 performs the processing of steps S1 to S8 to generate a super-resolution image of the next observation target surface. When determining that the imaging of the observation region in the depth direction has been completed (Step S9; Yes), the control unit 7 ends the series of processing.

In the above-described embodiment, the case where the sample S is irradiated with the activation light every time one modulated image is captured has been described. When the excitation light is applied, a part of the fluorescent substance may be in an inactivated state. Therefore, each time a modulated image is captured, the sample S is irradiated with the activation light, so that the fluorescent light inactivated by the excitation light is emitted. The substance can be activated again to take a modulated image.
For example, in the case where the direction of the interference fringe is changed in three ways and the phase of the interference fringe is changed in three ways in each direction of the interference fringe, the structured illumination microscope 1 uses nine different directions and phases of the interference fringe. A modulated image is captured in each of the combinations, and the activation light is applied to the sample S each time one modulated image is captured.

  The structured illumination microscope 1 may irradiate the sample S with activating light each time a plurality of modulated images are captured. For example, in the structured illumination microscope 1, the number of times of activating light irradiation processing may be one for the processing of capturing three modulated images while changing the phase of the interference fringes. Further, the structured illumination microscope 1 changes the combination of the direction and the phase of the interference fringe in nine ways, and performs one process of irradiating the activation light with one process for the process of capturing nine modulated images. There may be. Further, the structured illumination microscope 1 irradiates the sample S with the activation light by the activation illumination unit 3, irradiates the sample S with the excitation light by the excitation illumination unit 4, and captures the image of the sample S by the imaging unit 5. Good.

  Note that, here, a case where one sample S includes a plurality of observation target surfaces in one sample S has been described, but the number of observation target surfaces may be one. In this case, the fluorescent dye of the sample S does not need to be inactivated, and the inactivation illumination unit 20 can be omitted. The deactivating illumination unit 20 has the activation illumination unit 3 and the first illumination optical system 11 in common, but irradiates the sample with inactivation light from a different optical path from the first illumination optical system 11. You may. The deactivating light emitted by the deactivating illumination unit 20 does not have to be a sheet. The structured illumination microscope 1 may use the excitation illumination unit 4 as the inactivation illumination unit 20.

  In step S3, the control unit 7 stops the irradiation of the activation light based on the preset amount of the activation light, but detects the activation level of the fluorescent dye in the sample S and detects the detection level. The activation light irradiation may be stopped based on the result.

  FIG. 7 is a flowchart illustrating an example of a process for detecting the activation level of the fluorescent dye in the sample S. The control unit 7 blocks the + 1st-order diffracted light in the excitation illumination unit 4 in step S20. For example, a shutter is provided in the opening of the mask 45 shown in FIG. 2, and the control unit 7 closes the shutter so that the + 1st-order diffracted light is not irradiated on the sample S. In step S21, the control unit 7 controls the light source unit 30 to cause the excitation illumination unit 4 to irradiate the sample S with −1st-order diffracted light. In this case, since the + 1st-order diffracted light is blocked, no interference fringes are formed on the sample S, and the excitation illumination unit 4 can illuminate the sample S with a uniform brightness as compared with the case where the + 1st-order diffracted light is not blocked.

  The control unit 7 causes the imaging device 56 to detect the fluorescence from the sample S in step S22, for example, while continuing to irradiate the sample S with the excitation light. In step S23, the control unit 7 determines whether or not the amount of time change of the detection value of the image sensor 56 is equal to or smaller than a threshold. The fact that the amount of time change of the detection value of the imaging unit 5 is equal to or less than the threshold value corresponds to the fact that the activation level of the fluorescent dye in the sample S is almost saturated. When the control unit 7 determines that the amount of time change of the detection value of the image sensor 56 is larger than the threshold value (Step S23; No), the processing of Step S22 and Step S23 is repeated. When the control unit 7 determines that the time change amount of the detection value of the imaging element 56 is equal to or smaller than the threshold (Step S23; Yes), the control unit 7 stops the activation light irradiation in Step S3. As described above, the control unit 7 irradiates the activation light until the activation state of the fluorescent substance is determined to be saturated. In this case, the structured illumination microscope 1 can image the sample S in a state where the activation level of the fluorescent dye in the sample S is substantially saturated. Note that when the detection value of the imaging element 56 is equal to or greater than the threshold, the control unit 7 may determine that the activated state of the fluorescent substance is saturated, and may stop the irradiation of the activation light.

  The structured illumination microscope 1 may detect the activation level of the fluorescent dye in the sample S while illuminating the sample S with interference fringes. In this case, the process of step S20 may not be performed. Further, the structured illumination microscope 1 may irradiate the sample S with excitation light in a state where the activation level of the fluorescent dye in the sample S is not saturated, and may capture a modulated image of the sample S. For example, if the activation level of the fluorescent dye in the sample S is saturated, the brightness of the modulated image may be excessive. Further, for example, in order to reduce the influence of the activation light irradiation on the sample S, the activation light irradiation may be stopped before the activation level of the fluorescent dye is saturated. In such a case, it may be determined whether or not the detection value of the image sensor 56 is equal to or less than the threshold value, instead of or in addition to the determination process of step S23.

  In step S11 shown in FIG. 6, the control unit 7 stops the irradiation of the inactivating light based on the preset amount of the inactivating light. The level may be detected, and irradiation of the activation light may be stopped based on the detection result.

  FIG. 8 is a flowchart illustrating an example of a process for detecting the level of inactivation of the fluorescent dye in the sample S. The control unit 7 blocks the + 1st-order diffracted light in the excitation illumination unit 4 in step S25. In step S26, the control unit 7 causes the excitation illumination unit 4 to irradiate the sample S with -1st-order diffracted light. The processing of steps S25 and S26 may be the same as the processing of steps S20 and S21 shown in FIG.

  The control unit 7 causes the image sensor 56 to detect the fluorescence from the sample S in step S27, for example, while continuing to irradiate the sample S with the excitation light. The control unit 7 determines whether or not the amount of time change of the image sensor 56 is equal to or smaller than a threshold in Step S28. The fact that the amount of time change of the detection value of the image sensor 56 is equal to or smaller than the threshold value corresponds to the fact that the level of inactivation of the fluorescent dye in the sample S is almost saturated. When the control unit 7 determines that the time change amount of the detection value of the image sensor 56 is larger than the threshold value (Step S28; No), the processing of Step S27 and Step S28 is repeated. When the control unit 7 determines that the amount of time change of the detection value of the image sensor 56 is equal to or smaller than the threshold value (Step S28; Yes), it stops the irradiation of the inactivating light in Step S11. In this case, the structured illumination microscope 1 can capture a modulated image on the next observation target surface in a state where the level of inactivation of the fluorescent dye in the sample S is substantially saturated. Therefore, when the next observation target surface is irradiated with the excitation light, generation of fluorescence from the previous observation target surface can be suppressed. In step S28, the control unit 7 determines whether the detection value of the imaging element 56 is equal to or less than the threshold instead of determining whether the time change of the detection value of the imaging element 56 is equal to or less than the threshold. May be.

  The activation illumination unit 3 is not limited to the configuration of the above-described embodiment as long as it can selectively illuminate a sheet-shaped region of the sample S. Hereinafter, a modified example of the activation illumination unit 3 will be described.

  FIG. 9A is a perspective view showing the first illumination optical system 11 of the first modification, and FIG. 9B is a diagram showing an optical path of the first illumination optical system 11 of the first modification. The first illumination optical system 11 has a collimator lens 14 and a cylindrical lens 15 on the light emission side of the optical fiber 13, and has a configuration in which the slit 16 and the lens group 17 are omitted from the configuration shown in FIG. As described above, the activation illumination unit 3 does not need to perform beam shaping by the slit 16, and in this case, the lens group 17 that projects the image of the slit 16 can be omitted.

  FIG. 10A is a perspective view illustrating a first illumination optical system 11 according to a second modification, and FIG. 10B is a diagram illustrating an optical path of the first illumination optical system 11 according to a second modification. The first illumination optical system 11 includes a collimator lens 14, a condenser lens 60, a pinhole 61, a collimator lens 62, and a cylindrical lens 15 from the optical fiber 13 side to the sample S side. The pinhole 61 has, for example, a circular opening. The condensing lens 60 condenses the activation light from the collimator lens 14 toward the opening of the pinhole 61. The collimator lens 62 collimates the activating light from the pinhole 61. The cylindrical lens 15 refracts the activating light from the collimator lens 62 on a plane parallel to the XZ plane, and hardly refracts the activating light on a plane parallel to the XY plane. The first illumination optical system 11 can also mold the activation light into a sheet. When the sheet-like activation is formed by using the cylindrical lens 15 as described above, the arrangement of the cylindrical lens 15 can be changed.

  FIG. 11A is a perspective view showing a first illumination optical system 11 of a third modification, and FIG. 11B is a diagram showing an optical path of the first illumination optical system 11 of the third modification. The first illumination optical system 11 includes a collimator lens 14, a slit 16, a collimator lens 62, and a lens 63 on the light emitting side of the optical fiber 13. The slit 16 has an opening 16a that is narrower than the beam diameter of the inactive light from the collimator lens 14 in the Z direction. Further, the slit 16 is disposed at a rear focal position of the collimator lens 62. The opening 16a (see FIG. 10D) has a shape in which the Z direction is the shorter direction of the Y direction and the Z direction (eg, a rectangular shape having short sides parallel to the Z direction). The collimator lens 62 and the lens 63 are arranged such that the front focal position of the lens 63 coincides with the rear focal position of the collimator lens 62, and the collimator lens 62 and the lens 63 illuminate the activation light passing through the opening 16 a of the slit 16 in a sheet shape. Focus on the area.

  FIG. 12A is a perspective view showing a first illumination optical system 11 of a fourth modification, and FIG. 12B is a diagram showing an optical path of the first illumination optical system 11 of a fourth modification. The first illumination optical system 11 includes a collimator lens 14, a slit 16, and a lens 63 on the light emission side of the optical fiber 13. The opening 16a of the slit 16 has a shape in which the Y direction is the shorter direction of the Y direction and the Z direction. The dimension of the opening 16a of the slit 16 is set smaller than the beam diameter of the activating light from the collimator lens 14 in the longitudinal direction (Z direction), for example. The slit 16 is disposed at a rear focal position of the lens 63. The lens 63 focuses the activating light passing through the opening 16a of the slit 16 on the illumination area. As described above, the first illumination optical system 11 may shape the activation light by the slit 16 and may not include the cylindrical lens.

  FIG. 13A is a perspective view illustrating a first illumination optical system 11 according to a fifth modification, and FIG. 13B is a diagram illustrating an optical path of the first illumination optical system 11 according to the fifth modification. The first illumination optical system 11 includes a collimator lens 14, a triangular prism 64 having an obtuse apex angle on the optical axis AX1, and a lens group 65 on the light emission side of the optical fiber 13. The triangular prism 64 has an axially symmetric shape with respect to the optical axis AX1 of the first illumination optical system 11. The triangular prism 64 condenses the activation light at two positions at the position of the pupil plane of the lens group 65 (the lenses 65a and 65b). When the condensed activating light is projected onto the sample by the lens 65b, the light projected on the sample becomes sheet light that is a Bessel beam in the Z direction. As described above, the first illumination optical system 11 irradiates the sample S with the sheet light as the Bessel beam in the Z direction as the activating light. Such a first illumination optical system 11 can increase the depth of focus and reduce the thickness of the illumination area. Further, the first illumination optical system 11 can reduce light loss as compared with a configuration in which a part of the activating light is shielded to perform beam shaping. In this case, it is preferable to increase the diameter of the emission end of the optical fiber, so that the light density of the Bessel beam illumination light in the Z direction on the optical axis can be increased.

  FIG. 14A is a perspective view illustrating a first illumination optical system 11 of a sixth modification, and FIG. 14B is a diagram illustrating an optical path of the first illumination optical system 11 of the sixth modification. The first illumination optical system 11 includes a collimator lens 14, a slit 16, and a lens 66 on the light emission side of the optical fiber 13. For example, each of the opening 16a and the opening 16b of the slit 16 is a through hole whose inner edge is circular and whose inner diameter is sufficiently larger than the wavelength. The opening 16a and the opening 16b are separated from the optical axis AX1 of the first illumination optical system 11, and are provided symmetrically with respect to the optical axis AX1. Such a first illumination optical system 11 can form sheet light that is a Bessel beam in the Z direction without using the triangular prism 64 described above.

  FIG. 15A is a perspective view showing a first illumination optical system 11 of a seventh modification, and FIG. 15B is a diagram showing an optical path of the first illumination optical system 11 of a seventh modification. The first illumination optical system 11 includes a collimator lens 14, a mirror 67, and a lens 63 on the light emission side of the optical fiber 13. The mirror 67 is arranged in an optical path between the collimator lens 14 and the lens 63. The mirror 67 is provided rotatable about an axis parallel to the Z direction. The mirror 67 is driven by a mirror driving unit 68 to rotate. The mirror 67 and the mirror driving unit 68 are, for example, galvanometer mirrors, MEMS mirrors, and the like. The control section 7 can control the rotation position of the mirror 67 by controlling the mirror driving section 68. The activation light collimated by the collimator lens 14 is reflected by the mirror 67, is deflected in the Y direction according to the rotational position of the mirror 67, and is condensed by the lens 63. As described above, the activation illumination unit 3 may irradiate the activation light by scanning the sample S in the Y direction with the activation light (linear activation light) focused on the YZ plane.

  FIG. 16A is a perspective view illustrating a first illumination optical system 11 of an eighth modification, and FIG. 16B is a diagram illustrating an optical path of the first illumination optical system 11 of the eighth modification. The first illumination optical system 11 includes a collimator lens 14, a condenser lens 15, a pinhole 61, a collimator lens 62, a mirror 67, and a lens 63 on the light emitting side of the optical fiber 13. The condensing lens 15 condenses the activating light collimated by the collimator lens 14 toward the opening of the pinhole 61. The collimator lens 62 collimates the activating light from the pinhole 61. The mirror 67 deflects the activating light collimated by the collimator lens 62. The lens 63 focuses the activation light reflected by the mirror 67 on the illumination area. As described above, the first illumination optical system 11 may scan the sample S with the activation light (linear activation light) condensed on the YZ plane by forming the beam with the pinhole 61.

  FIG. 17A is a perspective view showing a first illumination optical system 11 of a ninth modification, and FIG. 17B is a diagram showing an optical path of the first illumination optical system 11 of the first modification. The first illumination optical system 11 includes a collimator lens 14, an axicon lens 69, a lens group 65, and a mirror 67 on the light emission side of the optical fiber 13. The axicon lens 69 and the lens group 65 shape the activation light collimated by the collimator lens 14 into a Bessel beam. The mirror 67 is arranged on the pupil plane of the lens group 65, and deflects the activation light passing through the lens group 65 in the Y direction. As described above, the first illumination optical system 11 may scan the sample S with the activation light formed into the Bessel beam using the axicon lens 69.

  FIG. 18A is a perspective view illustrating a first illumination optical system 11 according to a tenth modification, and FIG. 18B is a diagram illustrating an optical path of the first illumination optical system 11 according to the first modification. The first illumination optical system 11 includes a collimator lens 14, a slit 16, a lens group 65, and a mirror 67 on the light emission side of the optical fiber 13. The opening 16a of the slit 16 of the present modification has a ring shape (annular shape) centered on the optical axis AX1 of the first illumination optical system 11. The slit 16 and the lens group 65 shape the activation light collimated by the collimator lens 14 into a Bessel beam. The mirror 67 is arranged on the pupil plane of the lens group 65, and deflects the activation light passing through the lens group 65. As described above, the first illumination optical system 11 may scan the sample S with the activation light formed into the Bessel beam using the slit 16.

  FIG. 19 is a diagram illustrating the activation illumination unit 3 according to the eleventh modification. The activation illumination unit 3 of the present modification includes an optical path adjustment unit 70 that changes the optical path of the activation light in the Z direction. Note that FIG. 19A shows a state before the optical path of the activation light is changed, and FIG. 19B shows a state after the optical path of the activation light is changed.

  The optical path adjustment unit 70 includes, for example, a parallel flat plate 71 through which activation light passes, and a driving unit 72 that drives the parallel flat plate 71. The parallel flat plate 71 is arranged, for example, at a position where the activating light is parallelized in the optical path in the first illumination optical system 11. In this modified example, the parallel flat plate 71 is arranged in the optical path between the lens groups 17.

  The driving unit 72 drives the parallel flat plate 71 to rotate around an axis parallel to the Y direction, and changes the angle (inclination angle) of the parallel flat plate 71 with respect to the Z direction. The control unit 7 controls the angle of the parallel flat plate 71 with respect to the Z direction by controlling the driving unit 72. As shown in FIG. 19B, when the parallel plate 71 is inclined, the activation light is refracted at each of the interface on the incident side and the interface on the emission side of the parallel plate 71, and the optical path of the activation light is shifted in the Z direction. shift. The shift amount of the optical path of the activating light is determined by the refractive index of the parallel flat plate 71, the thickness of the parallel flat plate 71, and the inclination angle of the parallel flat plate 71. The control unit 7 can control the shift amount of the optical path by controlling the inclination angle of the parallel plate 71.

  Incidentally, the structured illumination microscope 1 can be used as an immersion type (oil immersion type) microscope in a state where an immersion liquid (eg, oil) is arranged between the objective lens 32 and the sample S. In this case, since the refractive index of the immersion liquid and the sample are generally different, when the stage 2 is moved in the Z direction while the objective lens 32 is fixed, the optical distance changes. For this reason, the high-contrast region of the structured illumination formed by the excitation illumination unit 4 and the focal plane of the objective lens 2 shift in the Z direction, and the illumination region of the activation illumination unit 3 and the illumination region in the Z direction are shifted. Misalignment. In such a case, the control unit 7 controls the driving unit 72 to shift the optical path of the activation light in the Z direction so that the illumination area of the activation illumination unit 3 matches the illumination area of the excitation illumination unit 4. be able to. The activation illumination unit 3 and the inactivation illumination unit 20 may be shifted in the Z direction relative to the excitation illumination unit 4 and the imaging unit 5.

  FIG. 20 is a diagram illustrating an activation illumination unit 3 according to a twelfth modification. In this modified example, the light source unit 30 of the excitation illumination unit 4 also serves as the light source unit 21 of the inactivation illumination unit 20. The deactivating illumination unit 20 includes a mirror 75 that can be inserted into and removed from the optical path between the light source unit 30 and the condenser lens 35. FIG. 20A illustrates a state in which the mirror 75 is retracted from the optical path between the light source unit 30 and the condenser lens 35 (hereinafter, referred to as a retracted state), and FIG. The state in which the mirror 75 is inserted in the optical path between the lens 75 and the condenser lens 35 (hereinafter referred to as an inserted state) is shown.

  As shown in FIG. 20A, in the retracted state of the mirror 75, the excitation light emitted from the light source unit 30 enters the condenser lens 35 and irradiates the sample S via the second illumination optical system 31. (See FIG. 1). Further, as shown in FIG. 20B, in a state where the mirror 75 is inserted, the excitation light emitted from the light source unit 30 is reflected by the wavelength selection film of the dichroic mirror 23 and enters the condenser lens 12. The excitation light incident on the condenser lens 12 is applied to the sample S as inactivating light via the first illumination optical system 11 (see FIG. 1).

  The control unit 7 controls a driving unit (not shown) of the mirror 75 and can switch between the retracted state and the inserted state of the mirror 75. The amount of excitation light when capturing a modulated image of the sample S is set to be smaller than the amount of inactivation light when inactivating the sample S, for example. The control unit 7 can also reduce the light emission amount of the light source unit 30 in the retracted state of the mirror 75 as compared with the inserted state of the mirror 75.

[Second embodiment]
Next, a second embodiment will be described. In the present embodiment, the same components as those in the above-described embodiment are denoted by the same reference numerals, and description thereof will be omitted or simplified. FIG. 21 is a diagram illustrating the structured illumination microscope 1 according to the present embodiment. The structured illumination microscope 1 has a 3D-SIM mode for generating a super-resolution image of the sample S in the thickness direction (Z direction).

  In the present embodiment, the mask 45 has an opening through which the zero-order diffracted light generated by the diffraction grating 39 passes. The excitation illumination unit 4 is a composite interference fringe in which an interference fringe of ± 1 order two light beams, an interference fringe of 0 order and +1 order light beams, and an interference fringe of 0 order and −1 order light beams are synthesized. Is formed in the illumination area. In the synthetic interference fringes, the light intensity periodically changes in a plane parallel to the XY plane in the illumination area, and also periodically changes in a plane parallel to the YZ plane.

  In the present embodiment, there are five types of modulation components to be superimposed on the modulation image: a secondary modulation component, a −1st modulation component, a 0th modulation component, a + 1st modulation component, and a + 2nd modulation component. The control unit 7 controls, for example, the rotation driving unit 42 to change the direction of the interference fringes to a plurality (for example, three types), and controls the parallel driving unit 43 for each direction of the interference fringes to control the interference fringes. While changing the phase into a plurality (for example, five types), the imaging unit 5 captures the modulated image in each phase. The demodulation unit 6 separates five types of modulation components using a plurality of (15 in the above example) modulated images, and generates a super-resolution image. For demodulation, "Super-Resolution Video Microscopy of Live Cells by Structured Illumination", Peter Kner, Bryant B. Chhun, Eric R. Griffis, Lukman Winoto, and Mats GL Gustafsson, NATURE METHODS Vol.6 NO.5 , pp.339-342, (2009), but is not limited to this method.

  The structured illumination microscope 1 according to the present embodiment can also execute the 2D-SIM mode described in the first embodiment by opening and closing the opening through which the 0th-order diffracted light passes in the mask 45. For example, the structured illumination microscope 1 can switch between the 2D-SIM mode and the 3D-SIM mode according to a user's command. The 2D-SIM mode is selected, for example, when the thickness of the illumination area of the activation illumination unit 3 is equal to or less than a threshold (eg, 1 μm or less). The 3D-SIM mode is selected, for example, when the thickness of the illumination area of the activation illumination unit 3 exceeds a threshold.

  Note that the technical scope of the present invention is not limited to the above-described embodiment or the modified example. For example, one or more of the requirements described in the above embodiments or modifications may be omitted. Further, the requirements described in the above embodiment or the modified examples can be appropriately combined. In addition, as far as permitted by law, the disclosure of all documents cited in each of the above-described embodiments and modified examples is incorporated into the description of the text.

DESCRIPTION OF SYMBOLS 1 ... Structured illumination microscope, 2 ... Stage, 3 ... Activation illumination part,
4 Excitation illumination unit 5 Imaging unit 6 Demodulation unit 7 Control unit S Sample 10 Light source unit 11 First illumination optical system Reference numeral 20: deactivating illumination unit, 21: light source unit, 30: light source unit 31: second illumination optical system, 55: imaging optical system

Claims (20)

A first illumination optical system that irradiates the sample with activation light for activating a fluorescent substance contained in the sample from a first direction;
A second illumination optical system having an objective lens, and irradiating the sample with excitation light for exciting the fluorescent substance from the second direction different from the first direction with interference fringes through the objective lens;
A control unit for controlling the direction and phase of the interference fringes,
An imaging optical system having the objective lens and forming an image of the sample irradiated with the interference fringes;
An imaging element that captures the image formed by the imaging optical system and generates an image;
Using a plurality of the images generated by the imaging device, a demodulation unit that generates an image of the sample,
Equipped with a,
The second illumination optical system includes:
A branch portion that branches the excitation light into a plurality of diffracted lights by diffraction,
A structured illumination microscope , comprising: a light-shielding portion capable of switching between a passing state and a blocking state of + 1st-order or -1st-order diffracted light among the plurality of diffracted lights . The structured illumination microscope according to claim 1, wherein the first illumination optical system irradiates the sheet with the activation light. The structured illumination microscope according to claim 1, wherein the first illumination optical system includes a cylindrical lens. The said 1st illumination optical system scans the said activating light in the 3rd direction different from the said 1st direction and the said 2nd direction. The Claim 1 characterized by the above-mentioned. Structured illumination microscope as described. The structure according to any one of claims 1 to 4, further comprising a third illumination optical system configured to irradiate the sample with inactivating light for inactivating the fluorescent substance. Illumination microscope. The structured illumination microscope according to claim 5, wherein a wavelength of the excitation light is the same as a wavelength of the inactivation light. The control unit includes:
Irradiating the activating light from the first direction to activate the fluorescent substance,
Irradiating the activated fluorescent substance with the excitation light from the second direction;
The structured illumination microscope according to claim 5, wherein the sample is irradiated with the inactivating light after the excitation light is irradiated. The structured illumination microscope according to any one of claims 5 to 7, wherein the third illumination optical system irradiates the sample with the inactivating light from a first direction. The structured illumination microscope according to any one of claims 5 to 7, wherein the third illumination optical system irradiates the sample with the inactivating light from a second direction. The structured illumination according to any one of claims 5 to 9, wherein an optical path of the first illumination optical system and an optical path of the third illumination optical system are at least partially common. microscope. The structured illumination according to any one of claims 5 to 9, wherein an optical path of the second illumination optical system and an optical path of the third illumination optical system are at least partially common. microscope. The structured illumination microscope according to any one of claims 5 to 11, wherein the intensity of the inactivating light is higher than the intensity of the excitation light. The first illumination optical system includes an aperture member disposed at a position optically conjugate with the sample,
The aperture width of the aperture member is set to be larger when the deactivating light passes through the first illumination optical system than when the activation light passes through the first illumination optical system. A structured illumination microscope according to any one of claims 5 to 12. The structuring according to any one of claims 5 to 13, wherein the control unit stops the irradiation of the inactivating light when a detection value of the imaging element is equal to or less than a threshold. Illumination microscope. The said control part stops irradiation of the said inactivation light, when the amount of time change of the detection value of the said image pick-up element is below a threshold. The method as described in any one of Claim 5 to 13 characterized by the above-mentioned. Structured illumination microscope as described.   The structured illumination microscope according to any one of claims 1 to 15, wherein the control unit irradiates the activation light until the activation state of the fluorescent substance is saturated. . Wherein, when the detected value of the image pickup element is not less than the threshold value, the structured illumination as claimed in any one of claims 16 claim 1, characterized in that stops irradiation of the activation light microscope. Wherein, when: the time variation of the detected value is the threshold value of the imaging device, according to claim claim 1, characterized in that stops irradiation of the activation light from the first illumination optical system The structured illumination microscope according to any one of Claims 16 to 16. The control unit sets the light blocking unit to the blocking state, and causes the imaging device to detect light from the sample while irradiating the activation light from the first illumination optical system. Item 19. The structured illumination microscope according to any one of items 1 to 18. Irradiating the sample with activation light for activating a fluorescent substance contained in the sample from a first direction;
Irradiating the sample with excitation light for exciting the fluorescent substance from the second direction different from the first direction with interference fringes through the objective lens by a second illumination optical system having an objective lens; ,
Controlling the direction and phase of the interference fringes;
Forming an image of the sample irradiated with the interference fringes by an imaging optical system having the objective lens;
Capturing the image to generate an image;
Using the plurality of images generated by the imaging, to generate an image of the sample,
By the second illumination optical system, the excitation light is split into a plurality of diffracted lights by diffraction, and among the plurality of diffracted lights, switching between a passing state and a blocking state of a + 1st order diffracted light or a −1st order diffracted light;
An observation method comprising:

Images